Efficacy of rifaximin against covert hepatic encephalopathy and hyperammonemia in Japanese patients

Covert hepatic encephalopathy (CHE) impairs patient quality of life and occurs in approximately 30% of liver cirrhosis (LC) cases. Japanese clinical practice guidelines recommend rifaximin to treat overt HE (OHE). However, the usefulness of rifaximin against CHE is not thoroughly investigated in Japanese patients. We aimed to investigate the efficacy of rifaximin against hyperammonemia and CHE in Japan. We observed 102 patients with HE showing hyperammonemia secondary to LC and examined various biochemical and behavioral parameters following rifaximin treatment. CHE was diagnosed when the patients exhibited two or more abnormal neuropsychological test (NPT) scores but did not indicate OHE symptoms. In the 102 cases, a significant therapeutic effect of rifaximin on hyperammonemia was observed from 2 to 48 weeks after starting treatment. Excluding 10 patients diagnosed with OHE upon starting rifaximin treatment, 12 of the 92 remaining patients (11.8%) transitioned to OHE within 1 year. The 1 year cumulative OHE transition rate was 14.5%. Among the 24 patients with CHE diagnosed by the NPT for whom NPT results could be evaluated at 4 and 12 weeks after starting treatment, 10 (41.6%) had recovered from CHE at 12 weeks. When the factors contributing to recovery from CHE were examined by multivariate analysis, an ammonia level <129 μg/dL was a significant factor. Rifaximin was thus significantly effective against both hyperammonemia and CHE in Japanese patients.


Introduction
Hepatic encephalopathy (HE) is a neuropsychiatric syndrome caused by chronic liver conditions including cirrhosis, acute liver failure, and portosystemic shunts. Symptoms such as personality changes and sleep rhythm disorders may occur early. However, asterixis and impaired consciousness can manifest in the advanced stage, leading to coma and death [1,2]. The West Haven Criteria (WHC) [1] and International Society of Hepatic Encephalopathy (ISHEN) [3] classification systems are commonly used to classify HE progression. In the Liver Cirrhosis the therapeutic effect of rifaximin on CHE in patients who were diagnosed with CHE by NPT and agreed to NPT at 4 and 12 weeks after treatment with rifaximin.

Patient follow-up and additional treatment
The registered patients underwent blood tests before starting rifaximin treatment. Clinical symptoms, including asterixis, were evaluated. OHE was diagnosed according to the ISHEN criteria [3] and was defined by symptoms such as asterixis and disorientation. Background clinical data and the history of OHE were determined from interviews and clinical records. In cases where computed tomography data were available before starting treatment, muscle volume loss was determined by the simple method of examining the iliopsoas muscle at the third lumbar vertebra level using the Japanese Society of Hepatology criteria [13]. Additionally, the computed tomography data were used to determine the presence of a portosystemic shunt (�5 mm), and the maximum diameter was measured. In cohort 1, blood samples were tested for ammonia levels and other clinical parameters, and an evaluation of the presence of OHE was conducted at 2,4,8,12,24,36, and 48 weeks after starting treatment. Ammonia levels following the treatment were evaluated until rifaximin was discontinued, additional treatment for HE was initiated, or the dose of concomitant medications were increased to eliminate the effects of other treatments. Specifically, patients who received additional treatment or discontinued rifaximin were analyzed as censored. Additional treatment included the administration of synthetic disaccharides, other nonabsorbable antibiotics, branched-chain amino acids (BCAAs), zinc, and L-carnitine, as well as clinical procedures such as interventional shunt occlusion (such as balloon-occluded transfemoral obliteration) and partial splenic embolization. No additional nutritional counseling was provided for hyperammonemia during the observation period.

NPT and CHE diagnosis
In cohort 2, NPT were performed before treatment and at 4 and 12 weeks after treatment for patients who consented to further evaluation. The test was performed on a touch screen tablet using an application [8] developed by the Japanese Society of Hepatology. Medical staff assisted with the operation to eliminate the bias caused by user error. The cut-off score of each test was determined using the reference value for each age group, according to a previous report [8]. Among the tests included in the NPT, four tests (number connection tests A and B, digit symbol test, and block design test) were used to evaluate CHE. According to previous reports [1,5,14], CHE was diagnosed when the results of two or more of the NPT were abnormal and when the patient did not present symptoms that are characteristic of OHE, such as asterixis or disorientation. The proportion of patients with CHE before treatment and the proportion of patients who recovered from CHE after rifaximin administration were examined. Patients receiving additional treatment after the start of rifaximin were defined as not showing recovery from CHE, even if the NPT score improved over time.

Ethical considerations
The study protocol was approved by the institutional ethics committee of Hokkaido University (Trial Number: 017-0313) and conformed to the ethical guidelines of the Declaration of Helsinki. Written informed consent was obtained from all patients. The study was registered in the UMIN Clinical Trials Registry (UMIN000044146).

Statistical analysis
The Mann-Whitney U test was used to compare the data between two groups and analyze continuous variables. Fisher's exact test was used for the univariate analysis of categorical independent variables. Logistic regression and multivariate analyses were used for categorical independent variables. Receiver operating characteristic (ROC) curves were drawn to determine the optimal cut-off values for multivariate analysis. For the time course of a variable, the Wilcoxon signed-rank test was used for testing the significant difference between the two points. The rate of transition to OHE was analyzed using Gray's test. All statistical analyses were performed using EZR software [15].

Patient characteristics
Fig 1 shows the patient classification flowchart. Rifaximin administration was started for 119 patients with LC who exhibited hyperammonemia during the observation period. Within 2 weeks, 10 of these patients had died or were transferred to different hospitals. Treatment was discontinued for four patients within 2 weeks owing to side effects, as determined by the attending physician as follows: one developed constipation (grade 2), two developed a rash (grade 2), and one developed fatigue (grade 2). All patients recovered, and no other physical disorders or ailments were observed. Treatment of one patient was discontinued within 2 weeks following an assessment by the attending physician, who concluded that HE had improved in the patient. Additionally, two patients switched to rifaximin from other nonabsorbable antibiotics. Therefore, these 17 patients were excluded from further evaluation. We analyzed changes in the ammonia levels of the remaining 102 patients who received continuous rifaximin for 2 weeks or longer as cohort 1.

Transition to OHE and HE recurrence
Of the 92 patients without OHE at the initiation of rifaximin treatment in cohort 1, 12 (11.8%) transitioned to OHE within 12 months of treatment, with a 1-year cumulative OHE transition rate of 14.5% (Fig 3). In 18 cases (19.6%), additional treatment for hyperammonemia was administered at the attending physician's discretion, and a total of 30 patients (35.3%) required additional treatment for hyperammonemia or HE. The 1-year cumulative additional treatment rate was 29.4%.

Therapeutic effect of rifaximin on CHE
To investigate the effect of rifaximin on CHE, the results of the NPT before and after rifaximin administration were analyzed as cohort 2. Twenty-four patients diagnosed as having CHE by an NPT (before treatment) provided data for up to 12 weeks after starting treatment. Table 2 also shows the characteristics of these 24 patients. The average number of abnormal NPT scores was 2.9 before, 2.0 at 4 weeks after, and 1.8 at 12 weeks after treatment initiation. A significant decrease was observed at 4 and 12 weeks after treatment initiation (P < 0.01; Fig 4a). Ten patients (41.6%) did not meet the diagnostic criteria for CHE 3 months after treatment initiation, and the CHE complication rate was significantly reduced (P < 0.01; Fig 4b).

Predictive factors for recovery from CHE after rifaximin treatment
Next, we examined the factors involved in the recovery from CHE after rifaximin treatment.
In the univariate analysis, high ammonia levels and the concomitant use of synthetic disaccharide laxatives were the predictive factors for recovery from CHE, as indicated by significant differences (P < 0.05). Using ROC curve analysis, we obtained a cut-off value of 129 μg/dL for the blood ammonia level (Fig 5a). In the multivariate analysis using these factors, an ammonia level greater than 129 μg/dL was extracted as a factor that significantly predicted recovery from CHE (P = 0.04, Table 2). The recovery rate from CHE was 16.6% (2/12) in the group with an ammonia level <129 μg/dL and 66.7% (8/12) for the group with an ammonia level �129 μg/ dL. The CHE recovery rate was significantly higher in the latter group (P = 0.02; Fig 5b).

Discussion
We observed the prospective therapeutic effect of rifaximin in Japanese patients with LC and hyperammonemia. The ammonia level decreased significantly for 48 weeks after starting treatment. Additionally, approximately 40% of the patients exhibiting CHE before treatment did not meet the diagnostic criteria for CHE 3 months after starting treatment. In particular, recovery from CHE was likely to occur in cases with high blood ammonia levels before beginning treatment. Rifaximin has been used as a therapeutic agent for HE in Europe and the United States for many years, and there have been many reports of its therapeutic effect in patients in these countries. Rifaximin reportedly suppresses the recurrence of OHE and significantly reduces HE-related hospitalization compared with observations with a placebo [9]. In addition, rifaximin suppresses HE recurrence and increases the survival rate by reducing the gastroesophageal varix rupture rate, spontaneous bacterial peritonitis incidence, and hepatorenal syndrome incidence [17]. Therefore, rifaximin might help to effectively control LC complications other than HE. However, in Japan, rifaximin was approved quite recently; thus, data on the effectiveness of rifaximin in Japanese patients are still limited. Recently, Sezaki et al. reported a significant decrease in ammonia levels following rifaximin administration in Japanese patients with HE [18]. Kawaratani et al. [19] also reported the long-term efficacy of rifaximin in Japanese patients by analyzing ammonia levels in a multicenter study. Here, rifaximin administration significantly reduced blood ammonia levels (Fig 2). Therefore, rifaximin might be an effective treatment for OHE and hyperammonemia in Japanese patients. CHE has been attracting attention in recent years because it reduces patient quality of life and is associated with poor work capacity [20,21], poor driving ability [6,22], and sleep disorders [23]. There are various reports on the prevalence of CHE and MHE, with MHE prevalence ranging between 21 and 85% and CHE prevalence ranging between 35.8 and 50.9% among LC patients, with varying frequencies, depending on the population [5,14,[24][25][26][27][28][29][30]. According to reports from Japan, the prevalence of MHE is approximately 21% [14] or 30% [5].
Additionally, because CHE has been reported to transition to OHE at a consistent rate [24,29,31], it is important to treat it promptly. Although it is not recommended that all CHE cases  be treated following Japanese and the American Association for the Study of Liver Diseases/ European Association for the Study of the Liver clinical practice guidelines, additional treatment is proposed for high-risk patients, such as those with worsening background liver conditions. Miwa et al. reported a high transition rate from MHE to OHE in cases of zinc deficiency, high ammonia levels, high serum creatinine levels, and low platelet counts [32]. Since only patients receiving rifaximin were enrolled in this observational study, evaluating a transition to OHE in the absence of therapeutic intervention was not possible. However, since the rate of transition from CHE to OHE is reportedly high in patients with hyperammonemia [32], it is suggested that neuropsychological testing should be actively performed for at-risk patients to assess the presence of CHE, and in the case of a positive diagnosis, additional treatment should be considered.
Several reports have described the therapeutic effects of rifaximin on MHE [33][34][35][36]. In a randomized controlled trial comparing the effects of lactulose with those of rifaximin on MHE, recovery from MHE reportedly occurred in approximately 70% of the cases, with no significant difference observed between the effects of lactulose and those of rifaximin [34]. In Japan, it has been reported that number connection test-A and digit symbol test scores significantly improved 2 weeks after rifaximin administration in OHE cases [37]. However, to the best of our knowledge, the therapeutic effects of rifaximin on CHE/MHE in Japan have not been reported. We evaluated CHE using NPT and prospectively observed patients treated with rifaximin. Unlike the patients in the trial mentioned previously, more than 70% of the patients in this study were already prescribed other therapeutic agents (BCAAs and synthetic disaccharide laxatives) because BCAAs and synthetic disaccharide laxatives are recommended as standard treatments for HE and hyperammonemia, according to Japanese liver cirrhosis clinical practice guidelines [4]. However, there are a certain number of cases of intolerance owing to the problem of sweetness or diarrhea. Almost all patients who were unable to take synthetic disaccharide laxatives had these problems in this study. Because the concomitant rate of synthetic disaccharide laxative (such as lactulose) use in this study was similar to that in past clinical reports in Japan [18,19], our study is considered to reflect actual clinical practice. Even in cases wherein other therapeutic agents had already been administrated, rifaximin addition effectively ameliorated CHE in approximately 45% of all cases. Therefore, rifaximin administration appears to be a promising therapeutic agent for LC with CHE even when BCAAs and synthetic disaccharide laxatives have already been administered.
We also examined the characteristics of patients that are likely to show recovery from CHE. A high blood ammonia level before treatment was identified as a factor that significantly contributed to the recovery from CHE. Because high ammonia levels affect cognitive function [38,39] and many CHE diagnostic tools, such as NPT, measure cognition, higher baseline ammonia levels associated with CHE recovery likely resulted in improved NPT scores. Various diagnostic methods for CHE, such as NPT, are greatly affected by cognitive function, which was considered the reason for the influence of high ammonia levels on CHE recovery. The results of these diagnostic methods could be modified for patients with other potential psychiatric disorders and dementia. Here, patients with apparent clinical symptoms of psychiatric disorders, dementia, and pre-existing diseases were excluded; however, accurate diagnosis by imaging tests, such as magnetic resonance imaging of the brain, could not be performed. It is challenging to diagnose potential cases of dementia and psychiatric disorders; therefore, we could have included CHE cases and potential cases of dementia and psychiatric disorders. Moreover, the therapeutic effect on CHE might have been greater for those without these complications. Blood ammonia levels are relatively low in most cases of dementia and psychiatric disorders, and thus, the therapeutic effect of rifaximin might have been low.
Hyperammonemia is deeply involved in the development of HE [40]. However, it has also been reported that ammonia levels do not correlate linearly with the severity of HE [41]. In recent years, it has been reported that there are various mechanisms other than ammonia, such as inflammation, oxidative stress, increased bile acids, and lactate, that play a role in the onset of HE [42]. Therefore, it is possible that the effect of rifaximin is poor for CHE patients for whom the ammonia level is relatively low. As CHE cases with high ammonia levels can be expected to benefit from rifaximin treatment, additional treatment with rifaximin may suppress the transition to OHE and improve the quality of life of eligible patients.
Nonetheless, this study had some limitations. First, this was an observational study involving a single hospital and a small number of patients. Only 24 patients could be evaluated for CHE recovery using NPT. Second, CHE was evaluated only by NPT and was not confirmed by other evaluation methods. It would be advantageous to assess the psychometric hepatic encephalopathy score and perform the animal naming test and/or Stroop test. Third, dementia and other psychiatric disorders were excluded based on clinical symptoms, but strict diagnoses using imaging tests were not performed. The drugs associated with these disorders could not be examined. Fourth, the addition of or increase in the drugs affecting HE indirectly after the initiation of rifaximin was not examined. Finally, there were no restrictions on the co-administration of other drugs before or while starting rifaximin treatment, as this was an observational study. Therefore, our findings may not purely reflect the effectiveness of rifaximin. These limitations are expected to be addressed in future interventional studies using several CHE evaluation methods with a large number of cases. In conclusion, rifaximin had a positive effect on not only hyperammonemia but also CHE and therefore may be a promising therapeutic option.